Cytoarchitecture of habenulae in Atlantic salmon during smoltification. (A–D) and (E–H) show histological sections of the habenulae in parr upper mode and pre-smolt II respectively, following nuclear staining using Yo-Pro1 (purple) and IHC with an antibody directed against acetylated tubulin (green). (A–G) are horizontal sections, (D,H) are transverse sections. (A1–D1) and (H1–H7) are higher magnification views of the areas boxed in (A–D) and (H) respectively. Arrowheads in (D) and (H) delimit the ventral border of habenulae. Thin arrows in (B), (C-C1), (F) and (G) point towards ventricular cells harboring a pseudo-stratified neuroepithelium organization. Values in the bottom left corner of (E–F) refer section numbers from dorsal to ventral habenula levels in (A–C) and (E–G) and from anterior to posterior habenula levels in (D,H). The levels of horizontal sections are also shown by red lines in the scheme in the upper right panel. Abbreviations: FR, fasciculus retroflexus; HC, habenular commissure; L/R, Left/Right; Pi, pineal organ; Ppi, and parapineal organ. Scale bar = 200 µm.

Subdomain organization of habenulae in Atlantic salmon during smoltification. (A–R) respectively show sections of parr upper mode and pre-smolt II specimens following ISH with probes for Sskctd12b (A–C,J–L), Sskiss1 (D–F,M–O) and SsSox1b (G–I,P–R). (A–I) are horizontal sections, (J–R) transverse sections. Schemes in the upper line show the levels of the sections along the dorso-ventral (A–I) or antero-posterior (J–Q) axis as indicated. The numbers of the sections shown along these axes are also indicated in the bottom left corner of the photographs. (C1,F1,I1) and (K1,N1,Q1) show higher magnifications of the areas boxed in (C,F,I) and (K,N,Q). Dotted lines in (F1) and (N1) and in adjacent sections (C1,I1) and (K1,Q1) delineate the posterior (F1) and dorsal (N1) limits of Sskiss1 territory, which largely exclude Sskctd12b and Sssox1b signals. Black arrowheads in (A,J,K) point towards a left dorsal territory negative for SsKctd12b and expressing pax6 (see below). Scale bars = 200 µm.

Distinct cellular organizations in Sskctd12b, Sskiss1, Sssox1b habenular territories. (A–L) show horizontal sections of habenulae in a parr upper mode following IHC with an antibody directed against acetylated tubulin (green) and Yo-Pro1 staining (purple) (A–C), ISH with probes for Sskctd12b (D–F), Sskiss1 (G–I), Sssox1b (J–L). (A–L) are adjacent sections at respectively anterior, medial and posterior organ levels, approximately corresponding to those shown in Figures 2A–C. (A1), (B1), (C1) show higher magnifications of the areas boxed in (A), (B), (C) respectively. Numbers in the bottom left corner of each panel indicate section numbers along the dorso-ventral axis. A dotted line delimits the Sskiss1 posterior border in (B,C,E,F,H,I,K,L). White (A) or black (D,G,J) arrowheads point towards the dorsal kctd12b negative territory. Thin arrows point towards Sssox1b positive neural progenitors. (M,N,O) show alignments of Sskctd12b, Sskiss1 and Sssox1b territories based on adjacent sections. Same abbreviations as in Figure 1. Scale bars = 200 μm.

Pax6 expression in neural progenitors and a dorsal neuronal territory in the habenulae of Atlantic salmon. (A–H) show sections of a parr upper mode following IHC using antibodies directed against pax6 (A–C), PCNA (D–F) and following ISH using probes for Sssox1b (G) and Sskctd12b (H). (A,D,G) are transverse sections at the level indicated by dotted arrows in (B), (B–C,E,F,H) are horizontal sections at the level indicated by red lines on the front view of habenulae schematized in the bottom right panel. (A1–F1) are higher magnification views of the territories boxed in (A–F). Arrowheads point towards the left-restricted dorsal nucleus and a smaller, right-sided, dorsal population of clustered cells positive for pax6 but negative for PCNA. Thin arrows point towards posterior and ventral neural progenitors. Same abbreviations as in Figure 1. Scale bars = 200 μm.

3D reconstructions of habenulae. (A–C) show 3D reconstructions of habenulae respectively in a parr lower mode, parr upper mode and smolt, with rostral and caudal views on the left and on the right as indicated. The whole habenulae are shown in grey (first line), the dorsal habenulae (including the pax6 dorsal left nucleus) in light blue (second line), the ventral habenulae (comprising kiss1 and sox1b positive territories) in dark blue (third line), the posterior growth zone (PGZ) in purple (fourth line) and the pax6 left dorsal nucleus in green (fifth line). The habenular commissure is in black. A dotted vertical line indicates the midline. Abbreviations: Hb, habenulae; L, left; R, right. Scale bars = 150 μm.

Pattern of presence/absence of an asymmetric pax6 dorsal nucleus in actinopterygians. (A-R) Transverse sections of habenulae in the reedfish E. calabaricus [(A,B); 20 cm juvenile], spotted gar L. oculatus [(C,D); 8.5 cm juvenile], European eel A. anguilla [(E–H); yellow resident stage], silver arowana O. bicirrhosum [(I–L); 7 cm juvenile], zebrafish D. rerio [(M,P); adult] and false clownfish A. ocellaris [(Q–T); stage 6], following IHC with antibodies directed against pax6 (A,C,E,I,M,Q,G,K,O,S) and PCNA (B,D,F,J,N,R,H,L,P,T). (A1–H1), (Q1–T1) are higher magnification views of the territories boxed in (A–H), (Q–T). Arrows point towards pax6 and PCNA positive neural progenitors, white arrowheads point to left restricted pax6 positive and PCNA negative dorsal nuclei observed in the European eel and false clownfish as in the Atlantic salmon. Opened arrowheads indicate thalamic Pax6 positive neuronal populations. Scale bars = 100 μm in (A–L) and 50 μm in (M–T).

Subdomain organization of habenulae in the European eel. (A–M) transverse sections of habenulae in A. anguilla (silver specimens) following ISH with probes for Aakctd12b (A–D), Aakiss1 (E–H) and Aasox1 (I–M). The orientation of section planes is shown in a schematic lateral view of A. anguilla brain and section levels along the antero-posterior axis are shown as red dotted lines on a front view of habenulae schematized in the top right corner. (B1,F1,J1,D1,M1) are higher magnification views of the areas boxed in (B,F,J,D,M). Black arrowheads point towards the left dorsal nucleus positive for pax6, and negative for PCNA and Aakctd12b. The thin arrow in (M,M1) points towards Aasox1 positive neural progenitors. The dotted line in (F1) and adjacent sections (B1) and (J1) delimits the Aakiss1 positive territory, which is adjacent to, but largely excludes Aasox1 expression at anterior-most levels of the habenulae. At this level, Aakiss1 expands dorsally but it is restricted to the organ ventral parts at medial to posterior levels. Scale bars = 200 μm.